Name: practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay
Uploaded: 2018-03-12T13:00:00.000+00:00
Duration: 7 min 44 s
Description: Watch this Scientific Journal Video about Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay at JoVE.com

We would like to thank Dr. Arnulfo Mendoza who provided training in the PuMA technique. Additionally, we would like to acknowledge Drs. Chand Khanna, Susan Garfield (NCI/NIH), and Sam Aparicio (BC Cancer Agency) for providing use of their microscopes during the course of this study. This research was supported (in part) by the Intramural Research Program of the National Institutes of Health, Center for Cancer Research, Pediatric Oncology Branch. M.M.L. was supported by the National Institutes of Health Intramural Visiting Fellow Program (award 15335), and is currently supported by a Joan Parker Fellowship in Metastasis Research. P.H.S. is supported by British Columbia Cancer Foundation.

All animal protocols from which imaging data were obtained were performed with approval of the Animal Care and Use Committee of the National Cancer Institute, National Institutes of Health. All animal protocols discussed and portrayed in the article video have been approved by the University of British Columbia Animal Care Committee.
1. Preparation of tumor cells for injection and materials for the PuMA model
NOTE: The amount of solutions and cells will be enough for 1 mouse. Scale up as necessary if more mice are used in the study. For media recipes, refer to Table 1 and Table 2.
<ol>
	<li>Pre-warm 5 mL of A-Media in a 15 mL conical tube in a 37 oC water bath.</li>
	<li>Melt the 1.2% low melting agarose solution (in sterile water) using the lab microwave.</li>
	<li>Transfer 5 mL of the molten agarose into a 15 mL conical tube, and keep warm in a 37 oC water bath. Ensure the melted agarose is at 37 oC and liquid prior to the lung insufflation step.</li>
	<li>Pre-chill 30 mL of cell-culture grade PBS supplemented with 1X pen/strep in an ice bucket.</li>
	<li>In one well of a 6-well plate, pre-soak a gelatin sponge in 1.5 mL of B-media. The sponge will be the support anchor for the lung slices.</li>
	<li>Ensure the OS cells are about 70-90% confluent on the day of the procedure. Do not use cells that are over-confluent or if the media is orange to yellow since the cells are usually stressed and have diminished viability at this point. For the osteosarcoma cell lines, MNNG and HOS, 5 x 105 in a volume of 100 &#956;L will be used to inject into the tail vein of 1 mouse. Upscale accordingly if more mice are used for the study.</li>
	<li>Harvest the tumor cells using 0.25% trypsin-EDTA (3 mL for a T75 flask or 2 mL in a 10 cm culture dish). When cells are beginning to lift off the plate, neutralize the trypsin-EDTA with complete media. Spin down cells and rinse once with cell-culture grade PBS. Resuspend pellet in 5 mL of PBS.</li>
	<li>Perform a cell count by standard methods. It is best to make more cell suspension than what is actually needed since loss of cell suspension often occurs during needle draw-up and failed injection attempts.</li>
	<li>Perform a Trypan-Blue Exclusion Assay on a sample of the cell suspension to assess the viability of the cells. Proceed only if the cell show 90% viability or higher.</li>
	<li>Inject 5 x 105 cells in a volume of 100 &#956;L. To prepare an excess of 2X of this amount, 1 x 106 cells should be spun down and resuspended in 0.2 mL of HBSS.</li>
	<li>Place cell suspension in an ice bucket while preparing the mice for injection.</li>
</ol>
2. Tail Vein Injection and Lung Insufflation
NOTE: The amount of solutions and cells in this section will be enough for 1 mouse. Scale up as necessary if more mice are used in the study. For a list of the equipment, materials and surgical instruments used in the following steps, refer to Table 3 and Table 4.
<ol>
	<li>Warm a female mouse (age 6-8 weeks) under a heating lamp for 5 min&#160;in order to make their tail vein more visibly apparent. For murine K7M2 or K12 cells, use Balb/c mice; for human MG63.3, MG63, MNNG, and HOS cells, use severe combined immunodeficiency mice.</li>
	<li>Make sure the cell suspension is uniform by gently shaking the tube. Carefully draw up the cell suspension into the 1 mL syringe without the needle. Cap the syringe with a 27 gauge needle. Make sure the needle bevel is on the same side as the volume markings on the needle.</li>
	<li>Place the mouse in the restrainer, and swab the tail with an alcohol wipe.</li>
	<li>Proceed to perform a tail vein injection and inject the 100 &#956;L of the cell suspension. 5 min&#160;after the injection, place the mouse in a CO2 chamber and begin the euthanasia standard operating procedure (as outline by your institutional animal care committee). Cervical dislocation should not be used as a means of euthanasia since the procedure will damage the trachea.</li>
	<li>Once the mouse is euthanized, begin preparation of the laminar flow hood for insufflation of the mouse lung with the agarose/A-media solution. Within the laminar flow hood, set up a work area with your sterile pad. Onto this pad you will place your sterilized instruments, IV catheter, IV extension set, and gravity perfusion apparatus (see Supplemental Figure 1).</li>
	<li>Place the mouse in dorsal recumbancy. With sterile small scissors, carefully dissect out the sternum to expose the chest cavity. Take care to not puncture the lung since an agarose/A-media solution will be used to insufflate the lung.</li>
	<li>When dissecting out the sternum, dissect past the thoracic inlet on both sides the trachea. Expose the trachea by dissecting away the surrounding soft tissue.</li>
	<li>Cannulate the trachea with a 20 gauge IV catheter. Loosely tie a surgical knot using sterile catgut suture around the cannulated trachea.</li>
	<li>Attach an IV extension set from the catheterized trachea to the 10 mL syringe of the gravity perfusion apparatus.</li>
	<li>Combine the pre-warmed 37 oC agarose (5 mL) and A-media (5 mL) into a 1:1 mixture. Pour the liquid agarose/A-media solution into the 10 mL syringe of the gravity perfusion device. Prior to insufflation of lungs with agarose/A-media solution, ensure that the entire length of the extension set has been primed with agarose/A-media, thereby negating inadvertent insufflation of lung samples with air.</li>
	<li>Fill the lung the agarose/A-media solution until the lung is fully insufflated.</li>
	<li>Once the lung is fully insufflated, remove the cannula and firmly tie off the surgical knot to prevent leakage of the agarase/A-media solution through the trachea.</li>
	<li>Proceed to dissect out the pluck (trachea, heart, and lung) from the chest cavity. Take care to not puncture or damage the surface of the lung.</li>
	<li>Place the pluck (in no particular orientation) in the pre-chilled 30 mL of PBS supplemented with 1X pen/strep, and allow the agarose/A-media to solidify for 20 min.</li>
	<li>Using fine scissors and tweezers, cut small pieces of the lung (3 mm x 1.5 mm) as shown in Figure 1A. Smaller lung slices can be easily imaged using a 2.5X objective. Multiple slices can be cut per experimental condition, typically 4-10 slices per group. 
	NOTE: For each experimental group, place the lung slices into a separate well (6-well plate) containing a 2 x 2 cm gelatin sponge pre-soaked in B-media. The amount of media per well should be 1.5 mL. Change the media every 2-3 days. For drug studies, the frequency of changing the media/drug is user determined.</li>
</ol>
3. Widefield Fluorescence Imaging of Lung Slices and Analysis
NOTE: For widefield fluorescence imaging, smaller slices are cut (3 mm x 1.5 mm x 1 mm) in order to fit the lung section into 1 image using a 2.5X objective.
Image acquisition on a widefield fluorescence microscope:
<ol>
	<li>Lung slices are typically imaged at 0, 3, 7, and 14 days post-injection. In the sterile environment of the biological cabinet, carefully transfer the lung slices from the gelatin sponges to a sterile 35 mm glass-bottom round dish. Take care to dab off the excess fluid from the lung slice as the excess fluid will act as a mirror and reflect fluorescent light coming from the tumor cells.</li>
	<li>Arrange the lung slices in a similar manner depicted in Figure 1A. Each column would represent a different experimental condition. Take care to not cross-contaminate the lungs with the tweezers. Rinse with 70% ethanol and dry before handling lung slices from another experimental group.</li>
	<li>Optimize the imaging parameters (ie. gain, offset, exposure time, binning) that provides the best contrast between the fluorescent tumor cells and background lung tissue in the control (vehicle untreated) group. Use the same parameters from the control group to image the experimental groups. Save as tiff image format.</li>
	<li>For a scale reference, take a digital picture of a micrometer at the same objective.</li>
	<li>Since lung auto-fluorescence changes over time, the imaging parameters must be adjusted to the control lungs each imaging session. The imaging parameters for one session may not necessarily be optimal for subsequent imaging sessions.</li>
</ol>
Image Analysis: 
NOTE: The following image processing steps are done with ImageJ 1.51h software package 17.
<ol>
	<li>Open an image file in ImageJ (Figure 3A).</li>
	<li>Subtract background: Process &#62; Subtract Background &#62; Rolling ball radius (start with 50 pixels), uncheck &#34;Light Background&#34; (Figure 3B).</li>
	<li>Convert image to 8-bit format: Image &#62; Type &#62; 8-bit (Figure 3C).</li>
	<li>Set units to pixels: Image &#62; Enter &#34;Pixels&#34; in Unit of Length, enter 1 in &#34;Pixel width&#34;, &#34;Pixel Height&#34;, &#34;Voxel Depth&#34;. Check &#34;Global&#34; box to apply to subsequent images.</li>
	<li>Using the Polygon selection tool, outline the shape of the entire lung slice and determine the area (pixel2) of the total lung slice. This value will be used to calculate the percent tumor burden of the lung.</li>
	<li>Threshold image: Image &#62; Adjust &#62; Threshold &#62; highlight &#34;Default&#34; and &#34;B&#38;W&#34; &#62; use the slider to threshold the image such that the majority of tumor cells are accurately highlighted. Pressing &#34;Apply&#34; will result in a black and white image where the lung is all black, and the fluorescent lesions are white (Figure 3D). Pressing &#34;Apply&#34; a second time will invert the image where all fluorescent structures are now black shapes (Figure 3E).</li>
	<li>Quantification of the number and shape of lesions: 
	Analyze &#62; Set Measurements &#62; Check &#34;Area&#34;. Uncheck all other boxes. 
	Analyze Particles &#62; Size (pixel2): 0-infinity represents the range of shapes that ImageJ will enumerate. Empirically determine the smallest lesion that is deemed to be a single tumor cell in Day 0 vehicle images. Use the area of this single tumor cell as the lower limit for the remaining images in the data set. For the work presented in this paper, 11 pixel2 is set as the lower limit. For the work presented in the video example, 24 pixel2 is set as the lower limit. Leave &#34;Circularity&#34; range as 0-1. &#34;Show&#34; can be set to &#34;Nothing&#34;. Alternatively, if &#34;Show&#34; and &#34;Outlines&#34; is selected, a drawing of all outlined shapes is generated. Check &#34;Display results&#34; for a separate window of measurements to pop up. Optionally, having &#34;Add to manager&#34; box checked will save all the shapes quantified to a ROI manager, which can be saved for future reference. After pressing OK, a &#34;Results&#34; window will pop up containing all enumerated shapes and area measurements (Figure 3F).</li>
	<li>Copy and paste the data from the &#34;Results&#34; window into a spreadsheet. Use the SUM mathematical function in Excel to sum all the areas of the metastatic lesions for that particular lung slice. To assess the percent lung tumor burden of lung slice, divide the sum of the areas of metastatic lesions by the total area of the lung slice. This parameter is also known as Area fraction (AA) which is described further by Underwood 18. 
	Lung tumor burden = Sum of metastatic lesion areas/total area of lung slice</li>
	<li>Calculate the lung tumor burden for the rest of the lung sections in control group and remaining groups. Plot the average lung tumor burden per group over 0, 3, 7 and 14 days. Representative widefield fluorescent pictures of high and low metastatic human OS cells growing in the PuMA model at progressive time points are shown (Figure 4A). A line graph showing the fold-change (normalized to Day 0) in percent metastatic tumor burden over time is shown (Figure 4B).</li>
</ol>
4. Confocal Fluorescence Imaging of the PuMA Model
NOTE: For confocal imaging, tissue processing is similar to that in the previous section except that larger lung slices are cut (complete transverse sections, 1-2 mm thick) to allow for more ROIs to be imaged.
Labelling of lung parenchyma with DAR4M:
<ol>
	<li>Immerse lung slices in 10 &#956;&#956;M of DAR4M (in HBSS) for 45 min&#160;at 37 &#176;C. DAR4M labels reactive nitrogen species within the cells of the lung.</li>
	<li>After 45 min&#160;of incubation, rinse the lung slices with fresh HBSS.</li>
	<li>Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.</li>
	<li>The imaging parameters for the example images shown in Figure 5 are listed in Table 5.</li>
	<li>Acquire a Z-stack for a region of interest. Use the suggested Z slice thickness for Nyquist sampling. A representative movie of a 3D stack showing green fluorescent OS cells in the DAR4M-labeled lung tissue is provided in Movie 1 and Supplemental Movie 1.</li>
</ol>
For the example confocal images shown in Figure 5, the imaging session was terminal. If longitudinal imaging is required, the use of a 2-photon or multi-photon equipped confocal LSM microscope for imaging is advised since there is less photodamage to living tissues19,20. 
Imaging mito-RFP expressing MG63 cells in the PuMA model:
<ol>
	<li>Perform the PuMA protocol as outline in Step 1 and 2 with using MG63 cells expressing the mito-RFP construct.</li>
	<li>Place the lung slice in a 35 mm glass-bottom round dish, and image on the confocal microscope.</li>
	<li>The imaging parameters for the example images shown in Figure 6 are listed in Table 6. A representative movie of a 3D stack showing green fluorescent OS cells with red fluorescent mitochondria is provided in Movie 2 and Supplemental Movie 2.</li>
</ol>

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The authors have nothing to disclose.

The following technical article describes some practical aspects of the PuMA model in studying lung colonization in OS. Some critical steps in the protocol where researchers should take extra care include the following:
a) Cannulation of the trachea. The trachea can be easily damaged while dissecting the surrounding muscle and connective tissue. In addition, the needle of the catheter can easily be pushed through the trachea. Pay close attention to how the bevel of the needle enters the trache...

Improved outcomes for pediatric patients with metastatic osteosarcoma (OS) still remains a critical unmet clinical need 1. This underscores the importance of developing new molecularly-targeted therapies. Conventional chemotherapeutics that target tumor cell proliferation have not proven to be effective in treating metastatic disease, and thus novel strategies must target the metastatic process itself 2. The current article discusses the practical aspects of a relatively new type of ex vivo lung metastasis model, the pulmonary metastasis assay (PuMA) developed by Mendoza and colleagues3, which provides a useful tool in discovering new molecular drivers in lung metastasis progression in OS 4,5. Before proceeding, however, it would be prudent to briefly touch upon several current models of metastasis, and how the PuMA model offers several advantages over conventional in vitro assays.
Most experimental models used to study metastasis comprise of in vitro and in vivo systems that recapitulate either a specific step or several steps of the metastatic cascade. These steps include: 1) tumor cells migrating away from the primary tumor, 2) intravasation into nearby vessels (blood or lymphatic) and transit within circulation, 3) arrest at the secondary site, 4) extravasation and survival at the secondary site, 5) formation of micrometastases, and 6) growth into vascularized metastases (Figure 1). In vitro models of metastasis can include 2-dimensional (2D) migration and 3-dimensional (3D) Matrigel invasion assays which are reviewed in detail elsewhere 6. For in vivo models, the two commonly used model systems include: 1) the spontaneous metastasis model is where a tumor cells are orthotopically injected into a specific tissue type to form a local tumor which spontaneously sheds metastatic cells to distant sites; 2) the experimental metastasis model is where tumor cells are injected into the blood vessel upstream of the target organ. For example, a tail vein injection of tumor cells results in the development lung metastases5,7,8. Other experimental metastasis models include injection of tumor cells into the spleen or mesenteric vein which results in the development of liver metastases9,10. Practical considerations of these in vivo models are discussed in detail by Welch 11. Another in vivo model used to study metastasis in pediatric sarcomas is the renal kidney subcapsular tumor implantation model which results in local tumor formation and spontaneous metastasis to the lungs 12,13. A more technically demanding technique such as intravital videomicroscopy can directly visualize, in real-time, interactions between metastatic cancer cells and the microvasculature of a metastatic site (ie. lung or liver) as described by MacDonald14 and Entenberg15, or cancer cell extravasation in the chorioallantoic membrane as described by Kim 16.
The PuMA model is an ex vivo, lung tissue explant, closed culture system where the growth of fluorescent tumor cells can be longitudinally observed via fluorescence microscopy over a period of a month (see Figure 2A). This model recapitulates the initial stages of lung colonization (steps 3 to 5) in the metastatic cascade. Some major advantages of the PuMA model over conventional in vitro models are: 1) it provides an opportunity to longitudinally measure metastatic cancer cell growth in a 3D microenvironment that retains many features of the lung microenvironment in vivo 3; 2) PuMA allows the researcher to assess whether the knockdown of a candidate gene or drug treatment has anti-metastatic activity in the context of a 3D lung microenvironment; 3) the PuMA model is flexible with many types of fluorescence microscopy platforms (Figure 2B) such as widefield fluorescence microscopy or laser-scanning confocal microscopy, examples of each are shown in Figure 2C &#38; D, respectively. This article will discuss how to use the PuMA model to obtain longitudinal imaging data on the metastatic growth of enhanced green fluorescent protein (eGFP)-expressing, human high and low metastatic osteosarcoma cells (MNNG and HOS cells, respectively) using low-magnification widefield fluorescence. Examples of imaging a fluorescent dye which labels the lung parenchyma, and a red-fluorescent protein genetic reporter which labels mitochondria in OS cells in the PuMA model using confocal laser-scanning microscopy are also discussed.

<table><tbody><tr><td>&lt;strong&gt;Table 2&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Cell culture reagents for A-media, B-media, and complete media&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MNNG-HOS</td><td>ATCC</td><td>CRL-1547</td><td>highly metastatic OS cell line</td></tr><tr><td>HOS</td><td>ATCC</td><td>CRL-1543</td><td>poorly metastatic OS cell line</td></tr><tr><td>MG63.3</td><td>Amy LeBlanc Laboratory (NCI)</td><td>N/A</td><td>highly metastatic OS cell line</td></tr><tr><td>MG63</td><td>ATCC</td><td>CRL-1427</td><td>poorly metastatic OS cell line</td></tr><tr><td>10X M199 media</td><td>Thermofisher</td><td>11825015</td><td>Base media for A-media and B-media</td></tr><tr><td>Distilled Water (sterilized)</td><td>Thermofisher</td><td>15230-147</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>7.5% sodium bicarbonate solution</td><td>Thermofisher</td><td>25080094</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Hydrocortizone</td><td>Sigma-Alrich</td><td>H6909</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Retinol acetate-water soluable</td><td>Sigma-Alrich</td><td>R0635-5MG</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>Penicillin/Streptomycin 10X concentrated (10000 U/ml) solution</td><td>Thermofisher</td><td>15140122</td><td>Component of A-media &amp;amp; B-media, complete media.</td></tr><tr><td>Bovine insulin solution (10mg/ml)</td><td>Sigma-Alrich</td><td>I0516-5ML</td><td>Component of A-media &amp;amp; B-media</td></tr><tr><td>DMEM, high glucose</td><td>Thermofisher</td><td>11965092</td><td>Base media of Complete Media</td></tr><tr><td>L-Glutamine (200 mM)</td><td>Thermofisher</td><td>25030081</td><td>Component of Complete Media</td></tr><tr><td>Fetal Bovine Serum</td><td>Thermofisher</td><td>16000044</td><td>Component of Complete Media</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline</td><td>Thermofisher</td><td>14190144</td><td>Used in cell culture.</td></tr><tr><td>Hank&amp;rsquo;s Buffered Salts Solution, no calcium, no magnesium, no phenol red</td><td>Thermofisher</td><td>14175095</td><td>Used to resuspend cell pellet prior to injection</td></tr><tr><td>Trypsin-EDTA (0.25%), phenol red</td><td>Thermofisher</td><td>25200114</td><td>Used in cell culture.</td></tr><tr><td>DAR4M</td><td>Enzo</td><td>ALX-620-069-M001</td><td>Used to label lung parenchyma.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 3&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials for PuMA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zeiss 710 Confocal LSM</td><td>Zeiss</td><td>N/A</td><td>Upright LSM confocal microscope</td></tr><tr><td>Zeiss 780 Confocal LSM</td><td>Zeiss</td><td>N/A</td><td>Inverted LSM confocal microscope</td></tr><tr><td>SCID mice</td><td>Charles River</td><td>N/A</td><td>NOD.CB17-Prkdcscid/NcrCrl, female, age 6-8 weeks</td></tr><tr><td>GelFoam</td><td>Harvard Apparatus</td><td>59-9863</td><td>Used as a support for lung tissue sections.</td></tr><tr><td>SeaPlaque Agarose</td><td>Lonza</td><td>50100</td><td>Used during insufflation of the lung.</td></tr><tr><td>1 ml syringe with 27 gauge needle</td><td>Fisherscientific</td><td>14-826-87</td><td>Used for tail vein injection.</td></tr><tr><td>10 ml syringe</td><td>BD</td><td>309604</td><td>Used for insufflation of the lung.</td></tr><tr><td>20 gauge catheter</td><td>Terumo</td><td>SR-OX2032CA</td><td>Used during insufflation of the lung.</td></tr><tr><td>Abbott IV extension set (30&quot;, Sterile)</td><td>Medisca</td><td>8342</td><td>Used during insufflation of the lung.</td></tr><tr><td>Alcohol swabs</td><td>BD</td><td>326895</td><td>For wiping tail vein before injection</td></tr><tr><td>Sterile surgical gloves</td><td>Fisherscientific</td><td>Varies with size</td><td>Asceptic handing of mouse lungs</td></tr><tr><td>30 cm ruler</td><td>Staples</td><td></td><td>Used for insufflation of the lung.</td></tr><tr><td>Support stand for ruler</td><td>Pipette.com</td><td>HS29022A</td><td>Used for insufflation of the lung.</td></tr><tr><td>35 mm glass-bottomed culture dish</td><td>Ibidi</td><td>81158</td><td>Used during imaging of lung slices</td></tr><tr><td>Absorbent Underpads with Waterproof Moisture Barrier</td><td>VWR</td><td>56617-014</td><td>Used to line the sterile work area in the biological hood.</td></tr><tr><td>Catgut Plain Absorbable Suture</td><td>Braun</td><td>N/A</td><td>Used to tie off cannulated trachea.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Table 4&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Surgical instruments for PuMA&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Micro Dissecting Scissors 3.5&quot; Straight Sharp/Sharp</td><td>Roboz</td><td>RS-5910</td><td>For cutting lung sections</td></tr><tr><td>4&amp;rdquo; (10 cm) Long Serrated Straight Extra Delicate 0.5mm Tip</td><td>Roboz</td><td>RS-5132</td><td>For manipulating/holding lung sections.</td></tr><tr><td>4&amp;rdquo; (10 cm) Long Serrated Slight Curve 0.8mm Tip</td><td>Roboz</td><td>RS5135</td><td>For manipulating/holding lung sections.</td></tr><tr><td>Thumb Dressing Forceps; Serrated; Delicate; 4.5&quot; Length; 1.3 mm Tip Width</td><td>Roboz</td><td>RS-8120</td><td>For general dissection.</td></tr><tr><td>Thumb Dressing Forceps 4.5&quot; Serrated 2.2 mm Tip Width</td><td>Roboz</td><td>RS-8100</td><td>For general dissection.</td></tr><tr><td>Extra Fine Micro Dissecting Scissors 3.5&quot; Straight Sharp/Sharp, 20mm blade</td><td>Roboz</td><td>RS-5880</td><td>For general dissection.</td></tr><tr><td>Knapp Scissors; Straight; Sharp-Blunt; 27mm Blade Length; 4&quot; Overall Length</td><td>Roboz</td><td>RS-5960</td><td>For general dissection.</td></tr></tbody></table>

practical considerations in studying metastatic lung colonization in osteosarcoma using the pulmonary metastasis assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

Low-magnification widefield fluorescence microscopy
For widefield fluorescence microscopy of PuMA lung slices, representative images and quantification data are shown in Figure 2C, and Figure 4A and B. The metastatic propensities for high and low metastatic cell lines are visually apparent over progressive time points. MNNG cel...

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Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

practical-considerations-studying-metastatic-lung-colonization

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10.0K Views.  National Institutes of Health. The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope. 

Video: Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Medicine

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of micrometastases by radiologic imaging and the success in their therapeutic eradication remain limited.
While animal models have proven to be invaluable tools for cancer research1, the monitoring/visualization of micrometastases remains a challenge and inaccurate evaluation of metastatic spread in preclinical studies potentially leads to disappointing results in clinical trials2. Consequently, there is great interest in refining the methods to finally allow reproducible and reliable detection of metastases down to the single cell level in normal tissue. The main focus therefore is on techniques, which allow the detection of tumor cells in vivo, like micro-computer tomography (micro-CT), positron emission tomography (PET), bioluminescence or fluorescence imaging3,4. We are currently optimizing these techniques for in vivo monitoring of primary tumor growth and metastasis in different osteosarcoma models. Some of these techniques can also be used for ex vivo analysis of metastasis beside classical methods like qPCR5, FACS6 or different types of histological staining. As a benchmark, we have established in the present study the stable transfection or transduction of tumor cells with the lacZ gene encoding the bacterial enzyme &beta;-galactosidase that metabolizes the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) to an insoluble indigo blue dye7 and allows highly sensitive and selective histochemical blue staining of tumor cells in mouse tissue ex vivo down to the single cell level as shown here. This is a low-cost and not equipment-intensive tool, which allows precise validation of metastasis8 in studies assessing new anticancer therapies9-11. A limiting factor of X-gal staining is the low contrast to e.g. blood-related red staining of well vascularized tissues. In lung tissue this problem can be solved by in-situ lung perfusion, a technique that was recently established by Borsig et al.12 who perfused the lungs of mice under anesthesia to clear them from blood and to fix and embed them in-situ under inflation through the trachea. This method prevents also the collapse of the lung and thereby maintains the morphology of functional lung alveoli, which improves the quality of the tissue for histological analysis. In the present study, we describe a new protocol, which takes advantage of a combination of X-gal staining of lacZ-expressing tumor cells and in-situ perfusion and fixation of lung tissue. This refined protocol allows high-sensitivity detection of single metastatic cells in the lung and enabled us in a recent study to detect "dormant" lung micrometastases in a mouse model13, which was originally described to be non-metastatic14.

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

The novel protocol reported in the present study allows selective detection of lung metastases at single cell resolution in mice by combined in-situ lung perfusion and fixation and X-Gal staining of lacZ-tagged tumor cells.

Metastasis is the main cause of death in the majority of cancer types and consequently a main focus in cancer research. However, the detection of ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival rate of osteosarcoma patients with pulmonary metastasis is less than 30%.Therefore, the utilization of mouse models mimicking the osteosarcoma development in humans is of great significance for understanding the fundamental mechanism of osteosarcoma carcinogenesis and pulmonary metastasis to develop novel therapeutics. Here, detailed procedures are reported to generate the primary osteosarcoma and pulmonary metastasis mouse models via&#160;intratibia injection of osteosarcoma cells. Combined with the bioluminescence or X-ray live imaging system, these living mouse models are utilized to monitor and quantify osteosarcoma growth and metastasis. To establish this model, a basement membrane matrix containing osteosarcoma cells was loaded in a micro-volume syringe and injected into one tibia of each athymic mouse after being anesthetized. The mice were sacrificed when the primary osteosarcoma reached the size limitation in the IACUC-approved protocol. The legs bearing osteosarcoma and the lungs with metastasis lesions were separated. These models are characterized by a short incubation period, rapid growth, severe lesions, and sensitivity in monitoring the development of primary and pulmonary metastatic lesions. Therefore, these are ideal models for exploring the functions and mechanisms of specific factors in osteosarcoma carcinogenesis and pulmonary metastasis, the tumor microenvironment, and evaluating the therapeutic efficacy in vivo.

The present protocol describes intratibia osteosarcoma cell injection to generate mouse models bearing orthotopic osteosarcoma and pulmonary metastasis lesions.

Osteosarcoma is the most common primary bone cancer in children and adolescents, with lungs as the most common metastatic site. The five-year survival ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

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Lung Tumor Cell Recruitment Assay

Experimental Metastasis Assay

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Improved Visualization of Lung Metastases at Single Cell Resolution in Mice by Combined In-situ Perfusion of Lung Tissue and X-Gal Staining of lacZ-Tagged Tumor Cells

Models of Bone Metastasis

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

Intratibial Osteosarcoma Cell Injection to Generate Orthotopic Osteosarcoma and Lung Metastasis Mouse Models